To determine the sites of androgen and estrogen synthesis in the primate CL and whether changes in steroid hormone levels are related to steroidogenic enzyme expression, the enzymes converting progesterone to androgen (P450C17) and then to estrogen (P450arom) were detected by immunohistochemistry in macaque luteal tissue throughout the menstrual cycle and simulated early pregnancy. CL were collected from rhesus monkeys (n=3/group) in the early (d 2-4 post-LH surge), mid (d 6-8), mid-late (d 10-12), and late (d 14-15) luteal phase and after 1, 3, 6, or 9 days of hCG (Profasi, Serono) treatment beginning on d 9 of the luteal phase. Tissues were fixed in 10% neutral-buffered formalin, dehydrated and embedded in paraffin or embedded in OCT and fresh-frozen in liquid propane. Paraffin-embedded tissues were processed for antigen retrieval, then immunohistochemistry. Specific cytoplasmic staining for P450C17 and P450arom was present in luteal cells, but not in the microvasculature, within all luteal tissues examined. During the menstrual cycle, P450C17 stained luteal cells were located along the vascular tracts and around the periphery of the CL. Intensely-stained luteal cells were associated with blood vessels entering from the outer surface of the CL, not blood vessels returning from the CL centrum. Other luteal cells within the CL proper were lightly stained. In contrast, P450arom stained luteal cells were scattered throughout the luteal parenchyma, but not along the vascular tracts. Throughout the luteal phase, P450C17 and P450arom staining intensity were similar at all stages. In simulated early pregnancy, P450C17 immunostaining was not as easily distinguished due to darker nonspecific background staining of tissue sections. However, cells stained for P450C17 were present near blood vessels, with some positive cells scattered throughout the CL. P450arom immunostaining was heterogenous throughout the CL; many intensely stained cells were interspersed among others that were only lightly stained. Overall, cellular staining for P450arom increased from day 1 to day 9 of simulated early pregnancy. These results are consistent with a two-cell model for steroid hormone production in the primate CL, whereby theca-luteal cells produce androgen substrate which is converted to estrogens by granulosa-luteal cells.